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A Meyerhans

Publications and source records attributed to A Meyerhans.

At least 37 records · Page 2Linked to original sources
PubMed1996

Dynamics of viral variants in HIV-1 Nef and specific cytotoxic T lymphocytes in vivo.

The vigorous CTL response directed against HIV is considered to be important in reducing HIV viral load, although it is unable to stop ongoing viral replication, which generates new antigenic variants. We analyzed the impact of sequential changes in five epitopes of HIV-1 Nef on CTL recognition in four stable patients. A high rate of variation was found, and in all these patients we could detect CTL specific for 32 out of 36 autologous viral variants occurring in 5 HLA-A2- or HLA-B7-restricted Nef epitopes at two time points. Two distinct patterns for dynamics of CTL responses to viral variation were observed: 1) temporary amplification of viral variants followed by expansion of variant-specific CTL, ultimately leading to the disappearance of 12 out of the 14 initial epitope variants within two years. A second set of viral variants that had replaced the initial ones could also stimulate specific CTL precursors in the context of the same or an alternative HLA molecule; and 2) persistence of 2 viral variants in relatively conserved epitopes despite specific CTL recognition. Therefore, a remarkable flexibility of the immune system allows constant adaptation of CTL to multiple HIV variants and thus elimination of HIV variant-producing cells in slow progressors.

Amino Acid Sequence↗
PubMed1996

Human immune response to HIV-1 Nef. II. Induction of HIV-1/HIV-2 Nef cross-reactive cytotoxic T lymphocytes in peripheral blood lymphocytes of non-infected healthy individuals.

HIV-specific CD8+ cytotoxic T lymphocytes (CTL) are thought to have a beneficial role in HIV infection. In a previous report we have shown that HIV-1 Nef-specific CTL can be readily induced in peripheral blood lymphocytes of seronegative healthy young adults by in vitro stimulation with autologous Epstein-Barr virus-transformed B lymphoblastoid cell lines transfected with the HIV-1 nef gene. Here we demonstrate that these Nef-specific CTL can efficiently lyse HIV-infected primary CD4+ T lymphocytes. CTL of the blood donor tested were Nef-specific and restricted by the autologous MHC class I molecules HLA-A2 and HLA-B7. They recognized HIV-1 Nef in association with both restriction elements but HIV-2 Nef only in association with HLA-B7. The cross-reactivity of the induced effector cells together with the potent immunogenicity of Nef in healthy seronegatives further support the inclusion of Nef as a constituent of HIV vaccines.

B-Lymphocytes↗
PubMed1995

Sequence constraints and recognition by CTL of an HLA-B27-restricted HIV-1 gag epitope.

Previous studies on the variation of an immunodominant HLA-B27-restricted HIV-1 gag p24 epitope (KRWIIL GLNK, amino acids 263-272) have demonstrated the persistence of variants recognized by CTL. Sequence comparisons of HIV isolates showed that this region is relatively conserved and as a consequence might restrict antigenic variation. To evaluate the possibility of HIV-1 to yield infectious mutants of this epitope that lack the ability to bind to HLA-B27 or escape HLA-B27-restricted CTL recognition, single-point mutations were constructed in the infectious molecular clone of HIV-1 Lai. Changes of arginine 264, the anchor amino acid for HLA-B27, to lysine or glycine resulted in infectious HIV-1 variants. The respective synthetic peptides showed reduced ability to sensitize target cells for CTL recognition and a corresponding loss of binding affinity to HLA-B27. In contrast, mutation of glycine 269 to lysine or glutamate abrogated HIV-1 infectivity. The corresponding peptides were able to bind to HLA-B27 but were not recognized by CTL. These data show that HIV-1 tolerates some genetic variation of the HLA-B27-restricted CTL epitope in gag p24 and that single-point mutations can alter quantitatively the immunologic properties. Further, it demonstrates that the mere nonrecognition of peptides derived from quasispecies analysis of small regions might simply correspond to nonviable virus variants and cannot be taken as evidence for CTL escape mutants. Together with the previously published data on the persistence of CTL epitopes, these results suggest that CTL do not play a major role in driving HIV-1 evolution in vivo.

Amino Acid Sequence↗
PubMed1995

Infection of cultured immature dendritic cells with human immunodeficiency virus type 1.

An in vitro culture system was developed that facilitates detailed studies of the interaction of Human Immunodeficiency Virus (HIV) with dendritic cells (DC). Cultured immature DC were generated from adherent peripheral blood mononuclear cells in the presence of GM-CSF and IL-4. These cells were non-adherent, non-phagocytic and had a veiled surface appearance. They expressed high levels of MHC class I and II proteins, CD1a, B7/BB1 and low levels of CD4, and were known to possess a potent soluble antigen presenting capacity. Upon infection with the HIV-1 strains Lai (lymphocytotropic) and BaL (monocytotropic), the viral RNA was reverse transcribed to complete DNA provirus. However the infection was non-productive as judged from measuring the activity of the virus encoded reverse transcriptase in the culture supernatant. Thus HIV infection was restricted at a step post entry.

Antigen Presentation↗
PubMed1995

Monocyte-derived cultured dendritic cells are susceptible to human immunodeficiency virus infection and transmit virus to resting T cells in the process of nominal antigen presentation.

The susceptibility of monocyte-derived cultured dendritic cells (DCs) to human immunodeficiency virus (HIV) infection and their role in viral transmission in the immune response were studied in detail. We observed that highly purified cultured DCs were infected with the T-tropic Lai strain of HIV type 1 (HIV-1Lai) via the CD4 receptor, and this was followed by formation of the complete provirus as detected by PCR. HIV mRNAs were transcribed at only low levels, and virus production was undectable; however, the addition of the purified protein derivative antigen of tuberculin and of autologous resting T cells to HIV-1Lai-infected DCs but not to HIV-1Lai-infected macrophages led to massive HIV transmission and production. These data suggest that the interaction of infected DCs with T cells during the normal immune response could play an important role in the activation and expansion of HIV.

Antigen Presentation↗
PubMed1994

G-->A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription.

The quasispecies model for RNA viruses predicts the existence of a replication error threshold beyond which there is a melting or total loss of sequence information. Retroviral G-->A hypermutation is probably an example. Here it is shown that G-->A transitions may occur in both GpG and GpA dinucleotide contexts. Transitions in GpG preferentially occur via base mispairing at the ends of runs of G residues, whereas G-->A transitions within GpA may result from temporary dislocation of the primer and template strands by a single base. The two circumstances may be related by the local dCTP substrate concentration. An in vitro elongation assay shows that primer/template dislocation is more frequent for the human immunodeficiency virus type 1 reverse transcriptase than for murine or avian retroviral enzymes. Taken together these data suggest that G-->A hypermutation is an example of induced mutation whereby the viral reverse transcriptase is forced into making errors by imbalances in the intracellular dCTP concentration.

Base Composition↗
PubMed1994

Human immune response to HIV-1-Nef. I. CD45RO- T lymphocytes of non-infected donors contain cytotoxic T lymphocyte precursors at high frequency.

The immune response of peripheral blood lymphocytes (PBL) of non-exposed human individuals to the Nef protein of HIV-1 was studied. Nef is a regulatory protein of HIV which is immediately expressed after infection and which seems to be important in the pathogenicity of HIV. Nef may therefore serve as a potential target for effective immunity against HIV infection. Epstein-Barr (EBV)-transformed lymphoblastoid B cell lines (LCL) were established from four healthy young seronegative adults and transfected with the Nef gene. These cells served as stimulator cells for autologous PBL in vitro and as target cells for CTL. CTL responses were readily generated against Nef-transfected LCL, consisting of Nef-specific and putative EBV-specific CTL. Nef-specific CTL were generated exclusively from CD8+ cells and were MHC class I restricted. Since a vigorous Nef-specific CTL response in non-infected individuals was unexpected, CTL precursor frequencies were determined by limiting dilution analyses in non-fractionated PBL and in PBL separated into the CD45RO- (naive) and CD45RO+ (memory) T cell populations. As expected, the putative EBV-specific CTL precursors were predominantly found in the CD45RO+ subset at frequencies typical for memory T cells. Nef-specific CTL precursors, in contrast, were found predominantly in the CD45RO- population, at even higher frequencies of approximately 1/1000-1/3000. Nef may thus display either an unusually high number of immunogenic peptides or a limited number of peptides presented in a very efficient way, so that many T cells including low affinity cells, would be triggered.

Adult↗
PubMed1994

Restriction and enhancement of human immunodeficiency virus type 1 replication by modulation of intracellular deoxynucleoside triphosphate pools.

Human immunodeficiency virus type 1 (HIV-1) replication is shown to be sensitive to the intracellular concentration of deoxynucleoside triphosphate substrates. Addition of thymidine to established cell lines resulted in a dramatic reduction of virus production. The effect could be substantially alleviated by addition of deoxycytidine, which, alone, enhanced viral titers by a factor of 2 to 3. Hydroxyurea treatment abolished HIV-1 replication in peripheral blood mononuclear cells and could be reversed by deoxyadenosine. These data show that HIV-1 replication occurs under suboptimal DNA precursor conditions.

CD4 Antigens↗
PubMed1993

A pattern search method for putative anchor residues in T cell epitopes.

The binding affinity between an antigenic peptide and its particular major histocompatibility complex (MHC) molecule seems to be largely determined by only a few residues. These residues have been called "anchors" because of their property of fitting into "pockets" inside the groove of the MHC molecule. To predict natural antigenic epitopes within a longer sequence, it therefore appears to be important to know the motif or pattern describing the anchors, i.e. the anchors amino acid residue preference and the distance between anchor residues. A large set of MHC class I-restricted peptides has been described. Peptide sequences vary in length and lack an obvious common sequence motif. For a list of peptides belonging to one type of MHC class I molecule, we describe a method to find the most prominent sequence motif with at least two anchor residues. Briefly, antigenic sequences are aligned, and two anchor positions are searched for, where all anchor residues share a high similarity. The alignments are scored according to the similarity of their anchor residues. We show that the motifs predicted for the MHC alleles A2.1, B27, Kb, Kd, Db are in substantial agreement with experimental data. We derive binding motifs for the MHC class I alleles HLA-A1, A11, B8, B14, H-2Ld and for the MHC class II alleles I-Ab and I-As. In some cases, higher scores were obtained by allowing a slight variation in the number of residues between anchors. Therefore, we support the view that the length of epitopes belonging to a particular class I MHC is not uniform. This method can be used to predict the natural short epitope inside longer antigenic peptides and to predict the epitopes anchor residues. Anchor motifs can be used to search for antigenic regions in sequences of infectious viruses, bacteria and parasites.

Amino Acid Sequence↗
PubMed1992

Strand specific PCR amplification of low copy number DNA.

A method for the amplification of a single DNA strand at low copy number is described. It is a wholly PCR based approach which involves an initial linear amplification of the target using a tagged strand specific primer. This is followed by classical PCR amplification of the progeny using a pair of primers, one specific for the sequence tagged onto the 5' end of the first round primer, the second specific for the target sequence. Given the protocol used the ratio of the two strands in the final amplification product was 50:1.

Base Sequence↗
PubMed1992

Absence of selection of HIV-1 variants in vivo based on transcription/transactivation during progression to AIDS.

The activity of the human immunodeficiency virus type 1 (HIV-1) transactivation protagonists tat and TAR has been analyzed from sequential primary material. The sequences were amplified from uncultured peripheral blood mononuclear cells. Despite fluctuations within the tat and TAR quasispecies there was no obvious selection for a variant encoding more powerful transactivation components either in vivo or ex vivo, indicating that this system is not exploited during disease progression. The basal levels of the natural promoters were, depending on the cell line, two- to fourfold higher than that of the reference promoter, itself derived from ex vivo adapted HIV-1 Lai.

Acquired Immunodeficiency Syndrome↗
PubMed1992

High-resolution structure of an HIV-1 quasispecies: identification of novel coding sequences.

OBJECTIVE: To characterize an HIV-1 quasispecies in vivo at high resolution (1%) in order to determine its genetic structure. METHODS: The first coding exon of tat was amplified by polymerase chain reaction from uncultured peripheral blood mononuclear cells of an HIV-1-infected patient. The products were cloned into M13mp18 RF, and 106 clones were sequenced. RESULTS: Thirty-one different Tat protein variants were found. Amongst these, five major forms with frequencies of 44, 11, 8 and 5% were identified. All of the remaining 26 sequences were unique, 15 of which were defective. Within the variant spectrum a small number of genomes encoded novel open reading frames, for example, a tat-vpu fusion product. CONCLUSION: Some of the myriad proviruses present in an individual harbour novel coding sequences. While these are probably of little importance for AIDS pathogenesis they emphasize the ability of HIV to explore a huge range of genetic configurations.

Amino Acid Sequence↗
PubMed1991

Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli.

Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.

Base Sequence↗
PubMed1991

In vivo persistence of a HIV-1-encoded HLA-B27-restricted cytotoxic T lymphocyte epitope despite specific in vitro reactivity.

A large number of human immunodeficiency virus type 1 (HIV-1) specific HLA-restricted cytotoxic T cell (CTL) epitopes have been mapped, including an HLA-B27-restricted immunodominant epitope within p25gag. Accordingly, this segment of the HIV-1 provirus was amplified by the polymerase chain reaction from DNA derived from fresh uncultured peripheral blood mononuclear cells (PBMC) of four HLA-B27 HIV-1-infected individuals. In all cases the majority of infected PBMC bore sequences encoding the HLA-B27-restricted peptide. CTL escape mutants had not accumulated in vivo 8 and 14 months later despite demonstrable CTL activity in vitro. These data emphasize the importance of silently infected lymphocytes in evading immune surveillance.

Amino Acid Sequence↗